1. Field of the Invention
The present invention relates to the technical field of microscopes.
2. Description of the Related Art
A fluorescence observation method using multiphoton excitation has been known as a method of fluorescence observation with a microscope.
With the multiphoton excitation, a fluorescent material is irradiated with light beams having a wavelength corresponding approximately to the integral multiple of the absorption wavelength simultaneously, inducing an excitation phenomenon that is equivalent to the excitation phenomenon generated by the original absorption wavelength. The multiphoton excitation phenomenon is called a nonlinear phenomenon, which occurs with a rate proportional to the square of the intensity of the excitation light in the case of, for example, two-photon excitation.
Meanwhile, the light density of an excitation light focused by an objective lens of a microscope decreases in inverse proportion to the square of the distance from the focal plane. In other words, the multiphoton excitation phenomenon in a microscope occurs only in the area very close to the focal point, and the fluorescence is emitted only from that area.
Thanks to the above characteristics of the multiphoton excitation, a multiphoton-excitation laser scanning microscope does not require a confocal pinhole on the detection side that is used in a normal confocal microscope for shutting out the emission of fluorescence in areas other than the focal plane. The multiphoton excitation also has an advantage that the discoloration by the fluorescence in a sample can be suppressed, since the excitation phenomenon occurs only on the focal plane.
Meanwhile, since the excitation light used for the multiphoton excitation has a longer wavelength than usual, it generally becomes a light beam in the infrared domain. Generally, a light with a longer wavelength is less prone to scatter (Rayleigh scatter). Therefore, the excitation light used in the multiphoton excitation has a characteristic that it reaches deeper in a specimen having a scattering property, such as a living specimen. This means that the use of multiphoton excitation enables the observation into a deep area in a living body that could not be attained with a normal visible light.
Thus, the fluorescence observation utilizing the multiphoton excitation in a microscope has now become very effective.
Similarly, in a microscope utilizing a Second-Harmonic Generation (SHG), a light having a half wavelength of the irradiation light is detected. Therefore, an SHG microscope also has advantages such as less influence from the Rayleigh scatter and less light invasion caused by the light to the sample.
The observation of a specimen using the above-described microscopes often involves a preparation of the specimen in advance, using another observation method. Particularly, when observing a specimen using the patch-clamp method, an electrode needs to be disposed accurately on a specific position in the specimen. Conventionally, in such a case, the multiphoton-excitation observation and SHG observation have been carried out, after attaching a patch clamp using observation methods utilizing differential interference contrast (DIC) or oblique illumination.
However, the preparation of a specimen using the DIC and oblique illumination has a significant problem.
The DIC observation requires a Nomarski prism or a Wollaston prism to be disposed on the image side of the objective lens. However, the laser entering the objective lens is to produce spots while it is spilt by the prism into two light fluxes that are slightly shifted sideways on the sample position, causing the degradation of resolution and decrease in brightness. For this reason, with the observation using the laser, these prisms need to be removed from the light path, and they need to be inserted into the path, with the DIC observation using a transmitted illumination light. However, when carrying out observation using the patch-clamp method, there have been problems such as the slight shake generated by these operations causing the detachment of the electrode.
In addition, transmitted-light DIC using the laser requires a linearly-polarized light with a high extinction ratio. However, the extinction ratio of the laser itself is low, and the optical system on the way to the objective lens breaks its polarization. Therefore, the light needs to be transmitted through a polarizer. Meanwhile, in the nonlinear microscopy observation method, the excitation efficiency is proportional to an n-th power of the laser intensity. Therefore, the loss of light intensity due to the polarizer causes decreases in the detection sensitivity and in the depth limitation in deep observation of a scattering substance.
Meanwhile, the contrast method using oblique illumination involves the oblique illumination of a transmitted-illumination light, the angle being provided by disposing a slit on a front focal position of the condenser lens. For this reason, the slit needs to be removed from the optical path, when performing the multiphoton-excitation observation or the SHG observation. This also leads to problems such as the slight shake generated by the insertion and removal of the slit causing the detachment of the electrode.